RSK2-regulated gene signature associates with ER+ breast cancer

We used CRISPR RSK2 knock-out MCF-7 cells to identify RSK2-regulated genes. Overall design: Wild type and RSK2-KO cells were serum starved for 12-15h and treated with EGF(10nM)/E2(1nM) for 8h before RNA purified. Library constructions were performed using TruSeq RNA Library Prep Kit (Illumina), and the sequencing was performed on an Illumina HiSeq 2500. CLC Workbench 10.0 (Qiagen) was used to align raw sequenced reads to the human reference genome hg38 (Genome Reference Consortium GRCh38) and to analyze the differential gene expression.

本研究采用成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats，CRISPR）介导的RSK2敲除MCF-7细胞，以鉴定受RSK2调控的基因。整体实验设计：将野生型与RSK2敲除（RSK2-KO）细胞进行12~15小时血清饥饿处理，随后以10nM表皮生长因子（Epidermal Growth Factor，EGF）与1nM雌二醇（Estradiol，E2）处理8小时，之后提取总RNA。采用Illumina公司的TruSeq RNA文库制备试剂盒完成文库构建，并使用Illumina HiSeq 2500平台开展测序。使用Qiagen公司的CLC基因组工作台10.0（CLC Workbench 10.0）将原始测序读段比对至人类参考基因组hg38（基因组参考联盟GRCh38），并进行差异基因表达分析。